A/T and G/C within double-stranded natural DNA form “exclusive” base pairs, respectively, via specific hydrogen bonds, respectively. However, the fact that natural nucleic acid has 4 kinds of bases (2 kinds of base pairs) provides a limitation to the chemical and physical variety of nucleic acid. If the genetic codes can be extended due to unnatural base pairs (artificial base pairs), it is beneficial to introduce various functional elements into desired positions into nucleic acids or proteins. So far, in the research of the unnatural base pairs, searches have been directed to those which could be antagonistic to natural base pairs in each process of replication, transcription and translation based on the combination utilizing the hydrogen bonds between the bases or the combination utilizing the hydrophobicity of the bases.
Inventors of the present invention have developed various kinds of unnatural base pairs which have a different hydrogen bond type compared to natural base pairs and can exclude a pairing with a natural base due to the steric hindrance. For example, 2-amino-6-dimethylaminopurine (x) and 2-amino-6-thienylpurine (s) introducing a bulky substituted group at position 6 of purine and pyridine-2-on (y) having a hydrogen atom at a site complementary to the bulky substituted group have been designed. When they have investigated an incorporation into DNA by Klenow fragment and an incorporation into RNA by T7 RNA polymerase regarding these x-y and s-y base pairs formation, the unnatural base pair s-y utilizing the steric hindrance showed an extremely high selectivity for transcription reaction, and a RNA, in which y is site-specifically incorporated at a site complementary to s within a template DNA, was able to be synthesized (for example, see Patent Document 1). And then, the genetic code was extended using this s-y base pair to create a new codon-anticodon pairs corresponding to the noncanonical amino acid, thereby they have succeeded in synthesizing a protein incorporating the noncanonical amino acid site-specifically in vitro (for example, see Non-Patent Document 1). However, its selectivity between unnatural base pairs in replication reaction has not been so high compared to that in transcription reaction.
Recently, inventors of the present invention have developed an unnatural base pair, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carboaldehyde (Pa), having high selectivity and incorporation efficiency for replication and transcription reactions based on the formation of base pairs due to hydrophobic interactions (for example, see Non-Patent Document 2). In the replication reaction, a self-pairing of Ds bases is suppressed by using γ-amidotriphosphate to form a selective base pair between Ds and Pa. On the other hand, in the transcription reaction, it is possible to synthesize RNA by standard T7 RNA polymerase reaction based on complementary property of the Ds-Pa base pair.
However, the conventional technique relating to these unnatural bases has been studied about a recognition of unnatural bases by DNA polymerase or RNA polymerase and also about a specific complementary property and a stability between unnatural base pairs, whereas studies about various factors to act on in a translation stage, for example, an influence onto interaction between tRNA and aminoacyl-tRNA synthetase have not been studied yet.
On the other hand, the genetic codes can be extended by assigning the noncanonical amino acid to nonsense codons that are UAA (ochre), UGA (opal) or UAG (amber). The suppressor tRNA (tRNAochre, tRNAopal, and tRNAamber) corresponding to these nonsense codons have nonsense anticodons, UUA, UCA and CUA, respectively, and they are used with a mutant aminoacyl-tRNA synthetase which can attach the noncanonical amino acid to these suppressor tRNA for an extension of the genetic codes.
For example, the phosphorylation at a side chain of serine in a protein is a post-translational modification, relating to many important biological phenomena such as signal transduction and the like. If phosphoserine can be introduced into a desired position in a protein by extending genetic codes, it will be a very important tool for study of a role of the phosphorylation at a serine residue of a protein. Phosphoseryl-tRNA synthetase (SepRS) is a noncanonical aaRS, which recognizes phosphoserine in several archaebacterium and ligates it to tRNACys with anticodon GCA. Inventors of the present invention have already analyzed the three-dimensional structure of SepRS/tRNACys/phosphoserine complex derived from Archaeroglobus fulgidus. And then, based on the structure of the complex, the SepRS was modified to recognize a mutant tRNACys having UCA and CUA at anticodon, thereby it was shown that phosphoserine can be introduced into nonsense codon using such a mutant SepRS/tRNA pair (for example, see Non-Patent Document 3).    [Patent Document 1] Japanese Patent No. 3893057    [Non-Patent Document 1] Hirao, I. et al., An unnatural pair for incorporating amino acid analogs into proteins, Nature Biotechnology, Vol. 20, pp. 177-182 (2002)    [Non-Patent Document 2] Hirao, I. et al., An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA, Nature Methods, Vol. 3, pp. 729-735 (2006)    [Non-Patent Document 3] Fukunaga, R. & Yokoyama, S., Structural insights into the first step of RNA-dependent cysteine biosynthesis in archaea, Nature Structural & Molecular Biology, Vol. 14, pp. 272-279, (2007)